Purification of alcohol dehydrogenase from bovine liver crude extract by dye-ligand affinity counter-current chromatography

Alcohol dehydrogenase (ADH) was extracted from a crude bovine liver homogenate by dye-ligand affinity counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (x-axis CPC). The purification was performed using two types of polymer phase systems composed of 4.4% polyethylene glycol (PEG) 8000-7.0% dextran T500-0.1 M potassium phosphate buffers and 16% PEG 1000-12.5% potassium phosphate buffers, both containing a procion red dye as an affinity ligand at various pH values. The best purification was achieved using the PEG 1000-potassium phosphate system at pH 7.3 containing 0.05% procion red as a ligand. The upper PEG-rich phase containing procion red was used as the stationary phase and a crude bovine liver homogenate was eluted with the potassium phosphate-rich lower phase at 0.5 ml/min. After elution of bovine liver proteins in the homogenate, ADH still retained in the stationary phase was collected from the column by eluting with the PEG 1000-rich upper phase. Collected fractions were analyzed by ADH enzymatic activity and by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to detect contaminant proteins in the ADH fractions. The ADH was purified directly from crude bovine liver extract within 6h with minimum loss of its enzymatic activity.     

Purification by affinity chromatography of 2,4-dienoyl-CoA reductases from bovine liver and Escherichia coli

1. Dye-ligand chromatography using immobilized Cibacron blue F3GA (blue Sepharose CL-6B) and Procion red HE3B (Matrex gel red A) as matrices and general ligand chromatography employing immobilized 2',5'-ADP (2',5'-ADP-Sepharose 4B) and immobilized 3',5'-ADP (3',5'-ADP-Agarose) were employed for purification of NADPH-dependent 2-enoyl-CoA reductase and 2,4-dienoyl-CoA reductase from bovine liver (formerly called 4-enoyl-CoA reductase [Kunau, W. H. and Dommes, P. (1978) Eur. J. Biochem. 91, 533-544], as well as 2,4-dienoyl-CoA reductase from Escherichia coli. 2. The NADPH-dependent 2-enoyl-CoA reductase from bovine liver mitochondria was separated from 2,4-dienoyl-CoA reductase by dye-ligand chromatography (Matrex gel red A/KCl gradient) as well as by general ligand affinity chromatography (2',5'-ADP-Sepharose 4B/NADP gradient). The enzyme was obtained in a highly purified form. 3. The NADPH-dependent 2,4-dienoyl-CoA reductase from bovine liver mitochondria was purified to homogeneity using blue Sepharose CL-6B, Matrex gel red A, and 2',5'-ADP-Sepharose 4B chromatography. 4. The bacterial 2,4-dienoyl-CoA reductase was completely purified by ion-exchange chromatography on DEAE-cellulose followed by a single affinity chromatography step employing 2',5'-ADP-Sepharose 4B and biospecific elution from the column with a substrate, trans,trans-2,4-decadienoyl-CoA. 5. The application of dye-ligand and general ligand affinity chromatography for purification of NADPH-dependent 2,4-dienoyl-CoA reductases taking part in the beta-oxidation of unsaturated fatty acids is discussed. It is concluded that making use of coenzyme specificity for binding and substrate specificity for elution is essential for obtaining homogeneous enzyme preparations.     

Purification of beta-glucosidase activities from bovine spleen by affinity chromatography

Bovine spleen β-d-glucosidase, glucosylceramide: β-d-glucosidase and glucosylsphingosine: β-d-glucosidase were purified by chromatography on a “gluconate” Sepharose column. Ten other lysosomal acid hydrolases, present in the preparation applied to the column, were absent from the glucosidase fraction.     

Purification of malic enzyme from bovine heart mitochondria by affinity chromatography

A simple two-step method for the purification of malic enzyme from bovine heart mitochondria in high yield is described. It consists of successive affinity chromatography steps on immobilized C⁸-(aminohexyl)-NADP and N⁶-(aminohexyl)-ADP. The molecular weight estimated by gel filtration of the homogeneous enzyme is 250,000 and the subunit molecular weight by SDS-polyacrylamide gel electrophoresis is 59,000.     

Purification of xanthine oxidase from bovine milk by affinity chromatography with a novel gel

Abstract

A new affinity gel was synthesized for the purification of xanthine oxidase (XO, EC 1.2.3.22) from bovine milk. The gel was prepared on a Sepharose 4B matrix on which a spacer arm based on l-tyrosine was covalently attached via CNBr activation, followed by reaction with the XO inhibitor p-aminobenzamidine. The elution conditions of affinity gel were determined at different pH values and ionic strengths. Maximum elution of XO was achieved at pH 9.0 and ionic strength around 0.4. The overall purification for XO was 1645-fold with 20.49% yield. SDS-PAGE of the enzyme indicates a single band with an apparent MW of 150?kDa. The gel provides a simple, rapid and effective useful for the purification of XO. Heat stability was determined on purified XO activity. Xanthine oxidase was preserved up to 70% with activity exposure of 60?°C and incubated for 60?min. These results indicated that the enzyme was heat stable.     

Purification of four gelatin-binding proteins from bovine seminal plasma by affinity chromatography

Bovine seminal plasma contains three similar acidic proteins, which we have previously designated as BSP-A1, BSP-A2, and BSP-A3. These proteins contain two homologous domains that are similar to type II structures present in the gelatin-binding domain of fibronectin. The present data have revealed that these proteins, like fibronectin, also form complexes with gelatin, a denatured collagen. Based on this property, a single step affinity purification method has been developed. In addition to these three proteins BSP-A1, -A2 and -A3, another protein with an apparent molecular weight of 30,000 dalton (named BSP-30-kDa) also bound to the gelatin-agarose column. Elution of these proteins from affinity columns using a linear gradient of either urea or arginine gave essentially the same pattern with a high yield of 90–95%. The purified proteins were homogeneous by SDS-polyacrylamide gel electrophoresis, amino acid composition and HPLC. Chromatography of bull seminal vesicular fluid also exhibited an elution pattern similar to that obtained for bull seminal plasma. The availability of these purified proteins should aid in understanding the physiology of these gelatin-binding proteins.     

Purification by affinity chromatography of the dicarboxylate carrier from bovine heart mitochondria

Submitochondrial particles were prepared from bovine heart mitochondria, solubilized with Triton X-114 in the presence of lipids and submitted to hydroxylapatite chromatography. The eluate obtained, containing a mixture of mitochondrial carriers, was processed further by affinity chromatography using as ligand p-aminophenylsuccinate coupled via a diazo bond to aminohexyl-Sepharose 4B. The activity of the dicarboxylate exchanger was measured after reconstitution into asolectin vesicles at each step of the purification procedure. All samples studied were found to display substrate and inhibitor specificity similar to those described for the dicarboxylate carrier in mitochondria. The specific activity of the final material eluted from the affinity column was found to be about 1000-times higher than that of the Triton X-114 extract of submitochondrial particles. SDS-polyacrylamide gel electrophoresis analysis of the affinity chromatography eluate showed the presence of only two polypeptides.     

Purification of jack bean meal beta-D-galactosidase by a new affinity adsorbent.

A simple procedure has been developed for the purification of jack bean beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) by affinity chromatography employing a new affinity adsorbent. The ligand 6-N-beta-(4-aminophenyl)-ethylamino-3-O-beta-D-galactopyranosyl-6-deoxy-L-gulitol was prepared by the reaction between lactose and beta-(4-aminophenyl)-ethylamine and was coupled to cyanogen bromide activated Sepharose 4B via the amino groups of the 4-aminophenyl moiety. This affinity gel resulted in a 111-fold purification of beta-D-galactosidase with a 64% recovery of the enzyme. With p-nitrophenyl-beta-D-galactopyranoside as the substrate the apparent Km and V values were 0.59 mM and 1.87 mumol/min per mg, respectively. The method for purification of beta-D-galactosidase may be applicable to other glycosidases depending upon the choice of specific di- or oligosaccharides of known structures to be used in the preparation of ligands.     

Purification of a soluble ATPase from rat liver mitochondria by AMP-Sepharose affinity chromatography.

ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity was shown in the soluble fraction of rat liver micochondria. Two molecular forms (ATPase 1 and 2) were isolated. ATPase 1 has already been studied. The present paper deals with the purification method of ATPase 2 which was achieved by the following steps: (NH4)2SO4 precipitation. DEAE-cellulose chromatography, hydroxyapatite chromatography, Sephadex G100 filtration and AMP-Sepharose affinity chromatography. The purified protein was characterized by bidimensional polyacrylamide gel electrophoresis. Molecular weight evaluated by SDS-polyacrylamide gel electrophoresis and Sephadex G100 gel filtration was found to be 61 500 +/- 3000.     

Purification of pigeon liver malic enzyme by affinity chromatography

A simple and rapid method for the purification of malic enzyme (EC 1.1.1.40) from pigeon liver is described. Malic enzyme in the crude tissue extract was partially purified by heat treatment, ammonium sulfate fractionation, and DEAE-cellulose chromatography. Final purification was achieved by affinity chromatography on immobilized N⁶-(6-aminohexyl)-adenosine 2′,5′-bisphosphate. Apparently homogeneous enzyme was obtained in 2 days with 54% yield.     

Purification of human liver glycoprotein sialyltransferase by affinity chromatography

A simple procedure has been developed for purifying solubilized human liver glycoprotein sialyltransferase (EC 2.4.99.1) 16 000-fold in 4–5% yield. The procedure involves two centrifugation steps, affinity chromatography of the ultrasupernatant fluid on cytidine diphosphate-hexanolamine-agarose followed by gel filtration on Sephadex G-150. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that the purified sialyltransferase preparation contains approximately equivalent amounts of three protein bands (with apparent molecular weights of 61 000, 63 000 and 70 000) and is highly purified if not homogeneous.     

Purification of rat liver phenylalanine hydroxylase by affinity chromatography

Rat liver phenylalanine hydroxylase has been purified to homogeneity on a totally synthesized affinity matrix. The affinity matrix consisted of a succinylated diaminodipropylamine arm linked to Sepharose-4B, to which the cofactor, 6,7-dimethyl-5,6,7,8-tetrahydropterin, was covalently linked. The pure enzyme was eluted with buffered 50% ethylene glycol, 1 m KCl in one step after the 50% ammonium sulfate fraction of the rat liver homogenate was applied to the affinity column. Specific activities ranging from 1.4 to 3.0 units/mg of protein were obtained. The enzyme has been shown to be homogeneous by: (i) discontinuous gel electrophoresis, and (ii) sodium dodecyl sulfate gel electrophoresis. The subunit molecular weight was determined by the same technique and was calculated to be between 51,000 and 55,000.     

Purification of bovine striatal dopamine D-2 receptor by affinity chromatography

Bovine striatal dopamine D-2 receptor has been purified approximately 2000-fold by affinity chromatography. The receptor, solubilized with cholic acid and sodium chloride, was adsorbed on haloperidol-linked Sepharose CL-6B and eluted with spiroperidol. The adsorption of receptor to the affinity matrix was biospecific as preincubation of the solubilized preparation with D-2 receptor agonists or antagonists blocked retention of receptor. The process also displayed stereoselectivity with respect to (+)- and (-)-butaclamol. Nondopaminergic agents such as mianserin and propranolol failed to exhibit any effect on the adsorption process. Elution of the receptor was also biospecific, as dopaminergic drugs were most effective (spiroperidol greater than haloperidol greater than dopamine) in eluting the bound receptor; whereas other agents, e.g. propranolol, mianserin, and acetic acid, were only slightly effective. One-cycle affinity purification resulted in a recovery of 12% of the original membrane-bound dopamine D-2 receptor with a specific activity of 169,600 fmol/mg of protein as assayed with [3H]spiroperidol binding. The order of potency of D-2 agonists (N-propylnorapomorphine greater than NO434 greater than apomorphine greater than dopamine) and antagonists (spiroperidol greater than (+)-butaclamol greater than domperidone) with the purified preparation was found to be similar to that of the solubilized dopamine D-2 receptor.     

Purification of nucleosidediphosphatase of rat liver by metal-chelate affinity chromatography

A procedure is presented for the purification of nucleosidediphosphatase (nucleosidediphosphate phosphohydrolase, EC 3.6.1.6) of rat liver by affinity chromatography using metal conjugated to epoxy-activated Sepharose 6B. The enzyme is eluted from the conjugate by a solution of L-histidine. The enzyme, when bound to metal-chelate gel, is active in a suspended form, suggesting that the catalytic site is different from the site that binds to the metal-chelate gels. Substrate specificity and Km value of the enzyme obtained are similar to those of the enzyme obtained from the same sources by a conventional procedure, indicating that the metal-chelate affinity chromatography does not bring about any substantial change in the catalytic properties.     

Purification of aldehyde dehydrogenase from rat liver mitochondria by alpha-cyanocinnamate affinity chromatography.

1. alpha-Cyano-4-hydroxycinnamate was coupled to Sepharose CL-4B activated with 1,2:3,4-bisepoxybutane. 2. The low-Km rat liver mitochondrial aldehyde dehydrogenase was specifically bound to this affinity medium, and could subsequently be eluted with alpha-cyano-4-hydroxycinnamate. 3. The enzyme purified in this manner had a subunit molecular mass of 55 kDa and a pI of approx. 6.5. A minor component of approx. 57 kDa was also present and had a significantly higher pI value; this may be the precursor for aldehyde dehydrogenase. 4. alpha-Cyanocinnamate and some related compounds were found to be uncompetitive inhibitors of the enzyme. 5. No cytosolic aldehyde dehydrogenase was bound to the affinity column, but a protein from a rat liver post-mitochondrial supernatant with a molecular mass of approx. 25 kDa was bound, and could be eluted subsequently with alpha-cyano-4-hydroxycinnamate.     

Purification of bovine liver rhodanese by low-pH column chromatography

We report a purification of bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) using column chromatography under conditions that take advantage of recent information regarding the structure and stability of this enzyme. At low pH (e.g., pH 4-6), rhodanese is stabilized against inactivation processes. By maintaining rhodanese at low pH, column chromatography, and especially ion-exchange chromatography, becomes practical, without loss of enzymatic activity. A purification method involving the sequential use of cation-exchange, size-exclusion, and hydrophobic-interaction chromatography was developed, and rhodanese was purified with good yield to electrophoretic purity and high specific activity. Previous methods for purifying bovine liver rhodanese employ repeated ammonium sulfate fractionations and crystallization of the rhodanese. In these methods, it is difficult to separate rhodanese from yellow-brown contaminants in the final stages of the procedures. Here, yellow-brown contaminants, which copurify with rhodanese on the first two columns, are completely resolved by hydrophobic interaction chromatography. This method can be readily scaled up, requires no special equipment, eliminates the variability inherent in previous methods, and is less dependent upon experience.     

Purification of vasoactive intestinal peptide receptor from porcine liver by a newly designed one-step affinity chromatography

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membrane using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The solubilized VIP receptor has been purified approximately 50,000-fold to apparent homogeneity by a one-step affinity chromatography using a newly designed VIP-polyacrylamide resin. The purified receptor bound 125I-VIP with a Kd of 22.3 +/- 0.7 nM and retained its peptide specificity toward VIP-related peptides. The specific activity of the purified receptor (16,400 pmol/mg of protein) was very close to the theoretical value (18,900 pmol/mg of protein) calculated assuming one binding site/protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified receptor revealed a single band with an Mr of 53,000 after either silver staining or radioiodination. Affinity labeling of the purified receptor with 125I-VIP using dithiobis(succinimidyl propionate) gave a single radioactive band, the labeling of which was completely inhibited by an excess of unlabeled VIP. In conclusion, an Mr 53,000 protein containing the VIP-binding site was purified to homogeneity by a one-step affinity chromatography using immobilized VIP.